The overall aim of this application is to determine the significance of self-derived peptides in positive selection of CD4+ T-cells in the thymus in vivo. In the thymus, thymocytes bearing receptors that can interact with self MHC/peptide complexes are selected to mature. Furthermore, thymocytes that interact too strongly with self MHC/peptide complexes are eliminated as potentially autoaggressive. The importance of self-derived peptides in negative selection in the thymus is indisputable. The role of peptides in the process of positive selection is not as clear as it is in negative selection. Currently, there are no data that show that positive selection on natural MHC/peptide ligands is primarily peptide specific. This application uses the recently established in vivo model, in which all class II MHC molecules (Ab) are occupied by a single peptide E(52-68) (Ep), to study the specificity of CD4+ T-cells selected on this MHC/peptide complex. Experiments in Specific Aim 1 are designed to determine the repertoire and specificity of CD4+ T-cells selected by a single class II MHC/peptide complex. In particular, the investigator will study the repertoire of CD4+ T-cells that were positively selected by a single class II MHC/peptide complex and negatively selected on the same class II MHC with wild-type peptides. The specific task of Aim 2 is to isolate TCR genes that encode TCRs with identified antigen-specificity, selected in vivo on the AbEp complex. These genes will allow him to construct a TCR transgenic mouse with TCR of known specificity for both selection (AbEp) and activation (AbPCC). Finally, this transgenic TCR will be tested in mice that express different amounts of the positively selected AbEp peptide ligand. Furthermore, it will be determined whether this transgenic TCR can be positively selected by other self-derived MHC/peptide complexes or on antigenic AbPCC complex. The knowledge gained from studies in this application will provide important insight into the selection processes of CD4+ T-cells in vivo, and may provide a basis for manipulation of the T-cell repertoire - an issue of great medical importance.